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Abstract:

The present invention relates to 2'-chloroacetylenyl-substituted
nucleoside derivatives of the general formula (I):
##STR00001##
As well as pharmaceutical compositions comprising such compounds and
methods to treat or prevent an HIV infection, HBV infection, HCV
infection or abnormal cellular proliferation, comprising administering
said compounds or compositions. In addition, the present invention
includes processes for the preparation of such compounds, and the related
β-D and β-L-nucleoside derivatives.

Claims:

1. A compound represented by Formula (I): ##STR01321## or the β-L
enantiomer thereof, or a pharmaceutically acceptable salt, ester,
stereoisomer, tautomer, solvate, prodrug, or combination thereof,
wherein: R1 is selected from the group consisting of: 1) hydrogen;
2) --CN; 3) halogen; 4) --N3; and 5) Substituted or unsubstituted
--C1-C8 alkyl; R2 and R4a are independently selected
from the group consisting of: 1) halogen; 2) --CN; 3) --N3; and 4)
OR6; where R6 is selected from the group consisting of:
hydrogen, hydroxy protecting group, --C(O)R7, --C(O)OR7, and
--C(O)NR8aR8b; wherein R7 is selected from the group
consisting of: substituted or unsubstituted --C1-C8 alkyl,
substituted or unsubstituted --C2-C8 alkenyl, substituted or
unsubstituted --C2-C8 alkynyl, substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, and substituted or
unsubstituted heterocyclic; R8a and R8b are each independently
selected from the group consisting of: hydrogen and R7; or
alternatively R8a and R8b taken together with the nitrogen atom
to which they are attached form a heterocyclic ring; R3 is R6;
or alternatively R2 is --OR6 and R3 and R6 together
form a group selected from: --C(Me)2-, --C(CH2)4--,
--CH(Ph)--, --CH(OMe)-- and --P(O)(OH)--; B is selected from the group
consisting of: substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, and substituted or unsubstituted heterocyclic;
preferably a heterocycle containing at least one nitrogen atom; R5a
and R5b are independently selected from the group consisting of: 1)
hydrogen; 2) substituted or unsubstituted --C1-C8 alkyl; 3)
substituted or unsubstituted --C2-C8 alkenyl; 4) substituted or
unsubstituted --C2-C8 alkynyl; 5) or R5a and R5b are
taken together with the carbon atom to which they are attached to form a
group selected from --C3-C8 cycloalkyl, --C3-C8
cycloalkenyl, or --C3-C8 cycloalkynyl; R5 is selected from
the group consisting of: 1) hydrogen; 2) R6; 3)
--P(O)(OR7a)(OR7b); wherein R7a and R7b are each
independently selected from the group consisting of a) hydrogen; b)
unsubstituted or substituted --C1-C8 alkyl; 4)
--P(O)(OR7a)--O--P(O)(OR7b)(OR7c); wherein R7a and
R7b are previously defined; R7c is selected from the group
consisting of a) hydrogen; b) unsubstituted or substituted
--C1-C8 alkyl; 5)
--P(O)(OR7a)--O--P(O)(OR7b)--O--P(O)(OR7c)(OR7d);
wherein R7a, R7b and R7c are previously defined; R7d
is selected from the group consisting of a) hydrogen; b) unsubstituted or
substituted --C1-C8 alkyl; 6) ##STR01322## where X is O or S;
R9 is R7 wherein R7 is previously defined; R10,
R11 and R12 are each independently selected from the group
consisting of: a) hydrogen; and b) unsubstituted or substituted
--C1-C8 alkyl; or R11 is hydrogen, and R12 and
R10 taken together with the nitrogen which R10 is attached to
form a heterocyclic ring; or R11 and R12 taken together with
the carbon which they are attached form a ring; R13 is hydrogen or
R7, wherein R7 is previously defined; and 7) ##STR01323##
where X is O or S; n is 1-4; R8a and R8b are as previously
defined; R14 is hydrogen or --(CO)--R7, wherein R7 is as
previously defined; Or, R5 and R3 are taken together to form
##STR01324## where X is O or S; and R6 is as previously defined.

22. A method of treating a hepatitis C viral infection in a subject in
need thereof comprising administering to the subject a cytochrome P450
monooxygenase inhibitor or a pharmaceutically acceptable salt thereof and
a compound as set forth in claim 1.

Description:

RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Application
No. 61/538,312, filed on Sep. 23, 2011. The entire teachings of the above
application(s) are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates generally to compounds and
pharmaceutical compositions useful as antiviral and antiproliferative
agents. Specifically, the present invention relates to nucleoside
derivatives with 2'-chloroacetylenyl substitution and methods for their
preparation and use.

BACKGROUND OF THE INVENTION

[0003] Synthetic nucleosides such as 5-iodouracil and 5-fluorouracil have
been used for the treatment of cancer for many years. Since the 1980's,
synthetic nucleosides have also been a focus of interest for the
treatment of HIV and hepatitis.

[0004] In 1981, acquired immune deficiency syndrome (AIDS) was identified
as a disease that severely compromises the human immune system and that
almost without exception leads to death. In 1983, the etiological cause
of AIDS was determined to be the human immunodeficiency virus (HIV). In
1985, it was reported that the synthetic nucleoside
3'-azido-3'-deoxythymidine (AZT) inhibits the replication of human
immunodeficiency virus. Since then, a number of other synthetic
nucleosides, including 2',3'-dideoxyinosine (DDI), 2',3'-dideoxycytidine
(DDC), and 2',3'-dideoxy-2',3'-didehydrothymidine (D4T), have been proven
to be effective against HIV. After cellular phosphorylation to the
5'-triphosphate by cellular kinases, these synthetic nucleosides are
incorporated into a growing strand of viral DNA, causing chain
termination due to the absence of the 3'-hydroxyl group. They can also
inhibit the viral enzyme reverse transcriptase.

[0005] The success of various synthetic nucleosides in inhibiting the
replication of HIV in vivo or in vitro has led a number of researchers to
design and test nucleosides that substitute a heteroatom for the carbon
atom at the 3'-position of the nucleoside. European Patent Publication
No. 0,337,713 and U.S. Pat. No. 5,041,449, assigned to BioChem Pharma,
Inc., disclose 2-substituted-4-substituted-1,3-dioxolanes that exhibit
antiviral activity. U.S. Pat. No. 5,047,407 and European Patent
Publication No. 0,382,526, also assigned to BioChem Pharma, Inc.,
disclose that a number of 2-substituted-5-substituted-1,3-oxathiolane
nucleosides have antiviral activity, and specifically report that
2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (referred to below as
BCH-189) has approximately the same activity against HIV as AZT, with
little toxicity.

[0006] It has also been disclosed that
cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane ("FTC") has
potent HIV activity. Schinazi, et al., "Selective Inhibition of Human
Immunodeficiency viruses by Racemates and Enantiomers of
cis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-Oxathiolane-5-yl]-Cytosine"
Antimicrobial Agents and Chemotherapy, November 1992, 2423-2431. See also
U.S. Pat. Nos. 5,210,085; 5,814,639; and 5,914,331.

[0007] Another virus that causes a serious human health problem is the
hepatitis B virus (referred to below as "HBV"). HBV is second only to
tobacco as a cause of human cancer. The mechanism by which HBV induces
cancer is unknown. It is postulated that it may directly trigger tumor
development, or indirectly trigger tumor development through chronic
inflammation, cirrhosis, and cell regeneration associated with the
infection.

[0008] After a two to six month incubation period in which the host is
unaware of the infection, HBV infection can lead to acute hepatitis and
liver damage, that causes abdominal pain, jaundice, and elevated blood
levels of certain enzymes. HBV can cause fulminant hepatitis, a rapidly
progressive, often fatal form of the disease in which massive sections of
the liver are destroyed.

[0009] Patients typically recover from acute hepatitis. In some patients,
however, high levels of viral antigen persist in the blood for an
extended or indefinite period, causing a chronic infection. Chronic
infections can lead to chronic persistent hepatitis. Patients infected
with chronic persistent HBV are most common in developing countries. By
mid-1991, there were approximately 225 million chronic carriers of HBV in
Asia alone, and worldwide, almost 300 million carriers. Chronic
persistent hepatitis can cause fatigue, cirrhosis of the liver, and
hepatocellular carcinoma, a primary liver cancer.

[0010] In western industrialized countries, high risk groups for HBV
infection include those in contact with HBV carriers or their blood
samples. The epidemiology of HBV is very similar to that of acquired
immune deficiency syndrome, which accounts for why HBV infection is
common among patients with AIDS or AIDS related complex. However, HBV is
more contagious than HIV.

[0011] Both FTC and 3TC exhibit activity against HBV. Furman, et al., "The
Anti-Hepatitis B Virus Activities, Cytotoxicities, and Anabolic Profiles
of the (-) and (+) Enantiomers of
cis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-oxathiolane-5-yl]-Cytosine"
Antimicrobial Agents and Chemotherapy, December 1992, pp. 2686-2692; and
Cheng, et al., Journal of Biological Chemistry, Volume 267(20), pp.
13938-13942 (1992). Other compounds that exhibit activity against HBV in
humans include Clevudine or CLV (L-FMAU) (Pharmasset, Inc. under license
from The University of Georgia Research Foundation and Yale University),
and L-dT and L-dC (Idenix Pharmaceuticals, Inc.).

[0012] HCV is the major causative agent for post-transfusion and for
sporadic non A, non B hepatitis (Alter, H. J. (1990) J. Gastro. Hepatol.
1:78-94; Dienstag, J. L. (1983) Gastro 85:439-462). Despite improved
screening, HCV still accounts for at least 25% of the acute viral
hepatitis in many countries (Alter, H. J. (1990) supra; Dienstag, J. L.
(1983) supra; Alter M. J. et al. (1990a) J.A.M.A. 264:2231-2235; Alter M.
J. et at (1992) N. Engl. J. Med. 327:1899-1905; Alter, M. J. et al.
(1990b) N. Engl. J. Med. 321:1494-1500). Infection by HCV is insidious in
a high proportion of chronically infected (and infectious) carriers who
may not experience clinical symptoms for many years. The high rate of
progression of acute infection to chronic infection (70-100%) and liver
disease (>50%), its world-wide distribution and lack of a vaccine make
HCV a significant cause of morbidity and mortality. Currently, there are
three types of interferon and a combination of interferon and ribavirin
used to treat hepatitis C. Selection of patients for treatment may be
determined by biochemical, virologic, and when necessary, liver biopsy
findings, rather than presence or absence of symptoms.

[0013] Interferon is given by injection, and may have a number of side
effects including flu-like symptoms including headaches, fever, fatigue,
loss of appetite, nausea, vomiting, depression and thinning of hair. It
may also interfere with the production of white blood cells and platelets
by depressing the bone marrow. Periodic blood tests are required to
monitor blood cells and platelets. Ribavirin can cause sudden, severe
anemia, and birth defects so women should avoid pregnancy while taking it
and for 6 months following treatment. The severity and type of side
effects differ for each individual. Treatment of children with HCV is not
currently approved but is under investigation. While 50-60% of patients
respond to treatment initially, lasting clearance of the virus occurs in
only about 10-40% of patients. Treatment may be prolonged and given a
second time to those who relapse after initial treatment. Re-treatment
with bioengineered consensus interferon alone results in elimination of
the virus in 58% of patients treated for one year. Side effects occur but
the medication is usually well tolerated. Combined therapy (interferon
and ribavirin) shows elimination of the virus in 47% after 6 months of
therapy. Side effects from both drugs may be prominent.

[0014] A tumor is an unregulated, disorganized proliferation of cell
growth. A tumor is malignant, or cancerous, if it has the properties of
invasiveness and metastasis. Invasiveness refers to the tendency of a
tumor to enter surrounding tissue, breaking through the basal laminas
that define the boundaries of the tissues, thereby often entering the
body's circulatory system. Metastasis refers to the tendency of a tumor
to migrate to other areas of the body and establish areas of
proliferation away from the site of initial appearance.

[0015] Cancer is now the second leading cause of death in the United
States. Over 8,000,000 persons in the United States have been diagnosed
with cancer, with 1,208,000 new diagnoses expected in 1994. Over 500,000
people die annually from the disease in this country.

[0016] Cancer is not fully understood on the molecular level. It is known
that exposure of a cell to a carcinogen such as certain viruses, certain
chemicals, or radiation, leads to DNA alteration that inactivates a
"suppressive" gene or activates an "oncogene." Suppressive genes are
growth regulatory genes, which upon mutation, can no longer control cell
growth. Oncogenes are initially normal genes (called prooncongenes) that
by mutation or altered context of expression become transforming genes.
The products of transforming genes cause inappropriate cell growth. More
than twenty different normal cellular genes can become oncongenes by
genetic alteration. Transformed cells differ from normal cells in many
ways, including cell morphology, cell-to-cell interactions, membrane
content, cytoskeletal structure, protein secretion, gene expression and
mortality (transformed cells can grow indefinitely).

[0017] All of the various cell types of the body can be transformed into
benign or malignant tumor cells. The most frequent tumor site is lung,
followed by colorectal, breast, prostate, bladder, pancreas and then
ovary. Other prevalent types of cancer include leukemia, central nervous
system cancers, including brain cancer, melanoma, lymphoma,
erythroleukemia, uterine cancer, and head and neck cancer.

[0018] Cancer is now primarily treated with one or a combination of three
means of therapies: surgery, radiation and chemotherapy. Surgery involves
the bulk removal of diseased tissue. While surgery is sometimes effective
in removing tumors located at certain sites, for example, in the breast,
colon and skin, it cannot be used in the treatment of tumors located in
other areas, such as the backbone, or in the treatment of disseminated
neoplastic conditions such as leukemia.

[0019] Chemotherapy involves the disruption of cell replication or cell
metabolism. It is used most often in the treatment of leukemia, as well
as breast, lung, and testicular cancer.

[0020] There are five major classes of chemotherapeutic agents currently
in use for the treatment of cancer: natural products and their
derivatives; anthacyclines; alkylating agents; antiproliferatives (also
called antimetabolites); and hormonal agents. Chemotherapeutic agents are
often referred to as antineoplastic agents.

[0021] The alkylating agents are believed to act by alkylating and
cross-linking guanine and possibly other bases in DNA, arresting cell
division. Typical alkylating agents include nitrogen mustards,
ethyleneimine compounds, alkyl sulfates, cisplatin and various
nitrosoureas. A disadvantage with these compounds is that they not only
attack malignant cells, but also other cells which are naturally
dividing, such as those of bone marrow, skin, gastrointestinal mucosa,
and fetal tissue.

[0022] Antimetabolites are typically reversible or irreversible enzyme
inhibitors, or compounds that otherwise interfere with the replication,
translation or transcription of nucleic acids.

[0023] Several synthetic nucleosides have been identified that exhibit
anticancer activity. A well known nucleoside derivative with strong
anticancer activity is 5-fluorouracil. 5-Fluorouracil has been used
clinically in the treatment of malignant tumors, including, for example,
carcinomas, sarcomas, skin cancer, cancer of the digestive organs, and
breast cancer. 5-Fluorouracil, however, causes serious adverse reactions
such as nausea, alopecia, diarrhea, stomatitis, leukocytic
thrombocytopenia, anorexia, pigmentation and edema. Derivatives of
5-fluorouracil with anti-cancer activity have been described in U.S. Pat.
No. 4,336,381, and in Japanese patent publication Nos. 50-50383,
50-50384, 50-64281, 51-146482, and 53-84981.

[0024] U.S. Pat. No. 4,000,137 discloses that the peroxidate oxidation
product of inosine, adenosine or cytidine with methanol or ethanol has
activity against lymphocytic leukemia.

[0025] Cytosine arabinoside (also referred to as Cytarabin, araC, and
Cytosar) is a nucleoside analog of deoxycytidine that was first
synthesized in 1950 and introduced into clinical medicine in 1963. It is
currently an important drug in the treatment of acute myeloid leukemia.
It is also active against acute lymphocytic leukemia, and to a lesser
extent, is useful in chronic myelocytic leukemia and non-Hodgkin's
lymphoma. The primary action of araC is inhibition of nuclear DNA
synthesis. Handschumacher, R. and Cheng, Y., "Purine and Pyrimidine
Antimetabolites" Cancer Medicine, Chapter XV-I, 3rd Edition, Edited by J.
Holland, et al., Lea and Febigol, publishers.

[0026] 5-Azacytidine is a cytidine analog that is primarily used in the
treatment of acute myelocytic leukemia and myelodysplastic syndrome.

[0027] 2-Fluoroadenosine-5'-phosphate (Fludara, also referred to as FaraA)
is one of the most active agents in the treatment of chronic lymphocytic
leukemia. The compound acts by inhibiting DNA synthesis. Treatment of
cells with F-araA is associated with the accumulation of cells at the
G1/S phase boundary and in S phase; thus, it is a cell cycle S
phase-specific drug. Incorporation of the active metabolite, F-araATP,
retards DNA chain elongation. F-araA is also a potent inhibitor of
ribonucleotide reductase, the key enzyme responsible for the formation of
dATP.

[0028] 2-Chlorodeoxyadenosine is useful in the treatment of low grade
B-cell neoplasms such as chronic lymphocytic leukemia, non-Hodgkins'
lymphoma, and hairy-cell leukemia.

[0029] In light of the fact that acquired immune deficiency syndrome,
AIDS-related complex, hepatitis B virus and hepatitis C virus have
reached epidemic levels worldwide, and have tragic effects on the
infected patient, there remains a strong need to provide new effective
pharmaceutical agents to treat these diseases that have low toxicity to
the host. Further, there is a need to provide new antiproliferative
agents.

[0030] Therefore, it is an object of the present invention to provide a
method and composition for the treatment of human patients or other host
animals infected with HIV.

[0031] It is another object of the present invention to provide a method
and composition for the treatment of human patients infected with
hepatitis B or C.

[0032] It is a further object of the present invention to provide new
antiproliferative agents.

[0033] It is still another object of the present invention to provide a
new process for the preparation of 2'-chloroacetylenyl-substituted
nucleoside derivatives of the present invention.

SUMMARY OF THE INVENTION

[0034] The present invention includes β-D and β-L-nucleoside
derivatives, pharmaceutical compositions comprising such compounds, as
well as methods to treat or prevent an HIV infection, HBV infection, HCV
infection or abnormal cellular proliferation, comprising administering
said compounds or compositions. In addition, the present invention
includes processes for the preparation of such compounds, and the related
β-D and β-L-nucleoside derivatives.

[0035] The compounds of the invention are 2'-chloroacetylenyl-substituted
nucleoside derivatives of the general formula (I):

##STR00002##

or the β-L enantiomer thereof, or a pharmaceutically acceptable
salt, ester, stereoisomer, tautomer, solvate, prodrug, or combination
thereof, wherein: R1 is selected from the group consisting of:

[0036] 1) hydrogen;

[0037] 2) --CN;

[0038] 3) halogen;

[0039] 4) --N3; and

[0040] 5) Substituted or unsubstituted --C1-C8 alkyl;

R2 and R4a are independently selected from the group consisting
of:

[0041] 1) halogen;

[0042] 2) --CN;

[0043] 3) --N3; and

[0044]
4) OR6; where R6 is selected from the group consisting of:
hydrogen, hydroxy protecting group, --C(O)R7, --C(O)OR7, and
--C(O)NR8aR8b; wherein R7 is selected from the group
consisting of: substituted or unsubstituted --C1-C8 alkyl,
substituted or unsubstituted --C2-C8 alkenyl, substituted or
unsubstituted --C2-C8 alkynyl, substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, and substituted or
unsubstituted heterocyclic; R8a and R8b are each independently
selected from the group consisting of: hydrogen and R7; or
alternatively R8a and R8b taken together with the nitrogen atom
to which they are attached form a heterocyclic ring; R3 is R6;
or alternatively R2 is --OR6 and R3 and R6 together
form a group selected from: --C(Me)2-, --C(CH2)4--,
--CH(Ph)--, --CH(OMe)-- and --P(O)(OH)--. B is selected from the group
consisting of: substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, and substituted or unsubstituted heterocyclic;
preferably a heterocycle containing at least one nitrogen atom. R5a
and R5b are independently selected from the group consisting of:

[0045] 1) hydrogen;

[0046] 2) substituted or unsubstituted
--C1-C8 alkyl;

[0047] 3) substituted or unsubstituted
--C2-C8 alkenyl;

[0048] 4) substituted or unsubstituted
--C2-C8 alkynyl; and

[0049] 5) or R5a and R5b are
taken together with the carbon atoms to which they are attached to form a
group selected from --C3-C8 cycloalkyl, --C3-C8
cycloalkenyl, or --C3-C8 cycloalkynyl. R5 is selected from
the group consisting of:

[0050] 1) hydrogen;

[0051] 2) R6;

[0052] 3)
--P(O)(OR7a)(OR7b); wherein R7a and R7b are each
independently selected from the group consisting of a) hydrogen; b)
unsubstituted or substituted --C1-C8 alkyl;

[0053] 4)
--P(O)(OR7a)--O--P(O)(OR7b)(OR7c); wherein R7a and
R7b are previously defined; R7c is selected from the group
consisting of a) hydrogen; b) unsubstituted or substituted
--C1-C8 alkyl;

[0055] where X is O or S; R9 is R7 wherein R7 is
previously defined; R10, R11 and R12 are each
independently selected from the group consisting of: a) hydrogen; and b)
unsubstituted or substituted --C1-C8 alkyl; or R11 is
hydrogen, R12 and R10 taken together with the nitrogen which
R10 is attached to form a heterocyclic ring; or R11 and
R12 taken together with the carbon which they are attached form a
ring, preferably a carbocyclic or heterocyclic ring, and more preferably
a cycloalkyl or cycloheteroalkyl ring; R13 is hydrogen or R7,
wherein R7 is previously defined; and

[0056] 7)

##STR00004##

[0056] where X is O or S; n is 1-4; R8a and R8b are as
previously defined; R14 is hydrogen or --(CO)--R7, wherein
R7 is as previously defined. Or, R5 and R3 are taken
together to form

##STR00005##

where X is O or S; and R6 is as previously defined.

[0057] In another embodiment, the present invention provides a
pharmaceutical composition comprising a therapeutically effective amount
of a compound or combination of compounds of the present invention, or a
pharmaceutically acceptable salt form, prodrug, salt of a prodrug,
stereoisomer, tautomer, solvate, or combination thereof, in combination
with a pharmaceutically acceptable carrier or excipient.

[0058] In yet another embodiment, the present invention provides a method
of inhibiting the replication of an RNA or DNA containing virus
comprising contacting said virus with a therapeutically effective amount
of a compound or a combination of compounds of the present invention, or
a pharmaceutically acceptable salt, prodrug, salt of a pro drug,
stereoisomer, tautomer, solvate, or combination thereof. Particularly,
this invention is directed to methods of inhibiting the replication of
HIV, HBV and HCV.

[0059] In still another embodiment, the present invention provides a
method of treating or preventing infection caused by an RNA or
DNA-containing virus comprising administering to a patient in need of
such treatment a therapeutically effective amount of a compound or
combination of compounds of the present invention, or a pharmaceutically
acceptable salt form, prodrug, salt of a prodrug, stereoisomer, or
tautomer, solvate, or combination thereof. Particularly, this invention
is directed to methods of treating or preventing infection caused by HIV,
HBV and HCV.

[0060] Yet another embodiment of the present invention provides the use of
a compound or combination of compounds of the present invention, or a
therapeutically acceptable salt form, prodrug, salt of a prodrug,
stereoisomer or tautomer, solvate, or combination thereof, as defined
hereinafter, in the preparation of a medicament for the treatment or
prevention of infection caused by RNA or DNA-containing virus,
specifically HIV, HBV and HCV.

DETAILED DESCRIPTION OF THE INVENTION

[0061] In a first embodiment of the present invention is a compound of
Formula (I) as illustrated above, or a pharmaceutically acceptable salt,
ester or prodrug thereof.

[0062] In a particular embodiment of the present invention is a β-D
2'-chloroacetylenyl substituted nucleoside derivatives represented by
formula (II), or its β-L enantiomer, or pharmaceutically acceptable
salt or prodrug thereof:

##STR00006##

wherein R1, R2, R4a, Rya, R5b, R3, B are as
previously defined.

[0063] Illustrative structures of formula (II) can be represented, but not
limited, by formula (II-1˜II-25) and the β-L enantiomers
thereof:

##STR00007## ##STR00008## ##STR00009## ##STR00010##

wherein B is as previously defined;

[0064] In another particular embodiment of the present invention is a
β-D 2'-chloroacetylenyl-substituted nucleoside diester derivatives
represented by formula (III), or its β-L enantiomer, or
pharmaceutically acceptable salt or prodrug thereof:

##STR00011##

wherein R1, R2, R5a, R5b, R7, and B are as
previously defined.

[0065] In another particular embodiment of the present invention is a
β-D 2'-chloroacetylenyl-substituted nucleoside phosphonamidate
derivative represented by formula (IV), or its β-L enantiomer, or a
pharmaceutically acceptable salt or prodrug thereof:

##STR00012##

wherein R1, R2, R5a, R5b, R3, R9, R10,
R11, R12, R13, and B are as previously defined. X is O or
S. Illustrative structures of formula (IV) can be represented, but not
limited, by formula (IV-1˜IV-12):

##STR00013## ##STR00014##

wherein R1, R2, R5a, R5b, and B are as previously
defined.

[0066] In another particular embodiment of the present invention is a
β-D 2'-chloroacetylenyl-substituted nucleoside phosphate derivative
represented by formula (V), or its β-L enantiomer, or a
pharmaceutically acceptable salt or prodrug thereof:

[0067] In still another particular embodiment of the present invention is
a β-D 2'-chloroacetylenyl-substituted nucleoside phosphate
derivative represented by formula (VI), or its β-L enantiomer, or a
pharmaceutically acceptable salt or prodrug thereof:

[0068] In yet another particular embodiment of the present invention is a
β-D 2'-chloroacetylenyl-substituted nucleoside derivative
represented by formulas (I) to (VI), or the β-L enantiomer, or a
pharmaceutically acceptable salt or prodrug thereof, with B at each
occurrence is an optionally substituted aryl, heteroaryl, or
heterocyclic; preferably a heterocycle moiety containing at least one
nitrogen, most preferably a pyrimidinyl, purinyl group or the like of the
general formula of (B1) and (B2):

##STR00017##

wherein:

[0069] Y is selected from a group consisting of: O, S,
NR8a, NC(O)R7, NC(O)OR7 and NC(O)NR8aR8b;

[0078] In one embodiment of the invention, the
2'-chloroacetylenyl-substituted nucleoside derivatives of the invention
are the isolated β-D or β-L isomer. In another embodiment of
the invention, the nucleoside derivative is in an enantiomeric mixture in
which the desired enantiomer is at least 95%, 98% or 99% free of its
enantiomer. In a preferred embodiment, the nucleoside derivatives are
enantiomerically enriched.

[0079] In one embodiment of the present invention, the compounds of the
formula (I) are in the β-D configuration. In an alternate embodiment
of the present invention, the compounds of formula (I) are in the
β-L configuration.

[0080] The nucleoside derivatives depicted above are in the β-D
configuration, however, it should be understood that the nucleoside
derivatives can be either in the β-L or β-D configuration.

[0081] The nucleoside derivatives of the present invention are
biologically active molecules that are useful in the treatment or
prophylaxis of viral infections, and in particular human immunodeficiency
virus (HIV) and/or hepatitis B virus (HBV) infection. The compounds are
also useful for the treatment of abnormal cellular proliferation,
including tumors and cancer. In another embodiment of the present
invention, any of the active compounds are useful in the treatment of
HCV. One can easily determine the spectrum of activity by evaluating the
compound in the assays described herein or with another confirmatory
assay.

[0082] For instance, in one embodiment the efficacy of the antiviral
compound is measured according to the concentration of compound necessary
to reduce the plaque number of the virus in vitro, according to methods
set forth more particularly herein, by 50% (i.e. the compound's
EC50). In preferred embodiments the compound exhibits an EC50
of less than 15 or preferably, less than 10 micromolar in vitro.

[0083] In another embodiment, for the treatment or prophylaxis of a viral
infection, and in particular an HIV, HCV or HBV infection, in a host, the
active compound or its derivative or salt can be administered in
combination or alternation with another antiviral agent, such as an
anti-HIV agent or anti-hepatitis agent, including those of the formula
above. Alternatively, for the treatment of abnormal cellular
proliferation, such as tumors and cancer, in a host, the active compound
or its derivative or salt can be administered in combination or
alternation with another antiproliferative agent, such as an
anti-neoplastic agent, including those of the formula above. In general,
in combination therapy, effective dosages of two or more agents are
administered together, whereas during alternation therapy, an effective
dosage of each agent is administered serially. The dosages will depend on
absorption, inactivation and excretion rates of the drug as well as other
factors known to those skilled in the art. It is to be noted that dosage
values will also vary with the severity of the condition to be
alleviated. It is to be further understood that for any particular
subject, specific dosage regimens and schedules should be adjusted over
time according to the individual need and the professional judgment of
the person administering or supervising the administration of the
compositions.

[0085] The present invention also provides a pharmaceutical composition
for the treatment and/or prophylaxis of a viral infection, and in
particular a HBV, HCV or HIV infection, in a host, preferably a human,
comprising a therapeutically effective amount of an active compound of
the present invention, optionally in a pharmaceutically acceptable
carrier.

[0086] The present invention also provides a pharmaceutical composition
for the treatment and/or prophylaxis of an abnormal cellular
proliferation, such as tumors and cancer, in a host, preferably a human,
comprising a therapeutically effective amount of an active compound of
the present invention, optionally in a pharmaceutically acceptable
carrier.

[0087] The present invention also provides a pharmaceutical composition
for the treatment and/or prophylaxis of a viral infection, and in
particular a HBV, HCV or HIV infection, in a host, preferably a human,
comprising a therapeutically effective amount of an active compound of
the present invention, in combination with one or more other effective
antiviral agent, and in particular an anti-HBV, anti-HCV or anti-HIV
agent, optionally in a pharmaceutically acceptable carrier.

[0088] The present invention also provides a pharmaceutical composition
for the treatment and/or prophylaxis of an abnormal cellular
proliferation, such as tumors and cancer, in a host, preferably a human,
comprising a therapeutically effective amount of an active compound of
the present invention, in combination with one or more other effective
antinroliferative agent, such as an antineoplastic agent, optionally in a
pharmaceutically acceptable carrier.

[0089] The present invention also provides a method for the treatment
and/or prophylaxis of a viral infection, and in particular a HBV, HCV or
HIV infection, in a host, preferably a human, comprising administering to
the host a therapeutically effective amount of an active compound of the
present invention, optionally in a pharmaceutically acceptable carrier.

[0090] The present invention also provides a method for the treatment
and/or prophylaxis of an abnormal cellular proliferation, such as tumors
and cancer, in a host, preferably a human, comprising administering to
the host a therapeutically effective amount of an active compound of the
present invention, optionally in a pharmaceutically acceptable carrier.

[0091] The present invention also provides a method for the treatment
and/or prophylaxis of a viral infection, and in particular a HBV, HCV or
HIV infection, in a host, preferably a human, comprising administering to
the host a therapeutically effective amount of an active compound of the
present invention, in combination and/or alternation with one or more
other effective antiviral agent, and in particular an anti-HBV, anti-HCV
or anti-HIV agent, optionally in a pharmaceutically acceptable carrier.

[0092] The present invention also provides a method for the treatment
and/or prophylaxis of an abnormal cellular proliferation, such as tumors
and cancer, in a host, preferably a human, comprising administering to
the host a therapeutically effective amount of an active compound of the
present invention, in combination and/or alternation with one or more
other effective antiproliferative agent, such as an antineoplastic agent,
optionally in a pharmaceutically acceptable carrier.

[0093] The present invention also provides a use of an active compound of
the present invention, optionally in a pharmaceutically acceptable
carrier, for the treatment and/or prophylaxis of a viral infection, and
in particular a HBV, HCV or HIV infection, in a host, preferably a human.

[0094] The present invention also provides a use of an active compound of
the present invention, optionally in a pharmaceutically acceptable
carrier, for the treatment and/or prophylaxis of an abnormal cellular
proliferation, such as tumors and cancer, in a host, preferably a human.

[0095] The present invention also provides a use of an active compound of
the present invention, in combination and/or alternation with one or more
other effective antiviral agent, and in particular an anti-HBV, anti-HCV
or anti-HIV agent, optionally in a pharmaceutically acceptable carrier,
for the treatment and/or prophylaxis of a viral infection, and in
particular a HBV, HCV or HIV infection, in a host, preferably a human.

[0096] The present invention also provides a use of an active compound of
the present invention, in combination and/or alternation with one or more
other effective antiproliferative agent, such as an antineoplastic agent,
optionally in a pharmaceutically acceptable carrier, for the treatment
and/or prophylaxis of an abnormal cellular proliferation, such as tumors
and cancer, in a host, preferably a human.

[0097] The present invention also provides a use of an active compound of
the present invention, optionally in a pharmaceutically acceptable
carrier, in the manufacture of a medicament for the treatment and/or
prophylaxis of a viral infection, and in particular a HBV, HCV or HIV
infection, in a host, preferably a human.

[0098] The present invention also provides the use of an active compound
of the present invention, optionally in a pharmaceutically acceptable
carrier, in the manufacture of a medicament for the treatment and/or
prophylaxis of an abnormal cellular proliferation, such as tumors and
cancer, in a host, preferably a human.

[0099] The present invention also provides the use of an active compound
of the present invention, in combination and/or alternation with one or
more other effective antiviral agent, and in particular an anti-HBV,
anti-HCV or anti-HIV agent, optionally in a pharmaceutically acceptable
carrier, in the manufacture of a medicament for the treatment and/or
prophylaxis of a viral infection, and in particular a HBV, HCV or HIV
infection, in a host, preferably a human.

[0100] The present invention also provides the use of an active compound
of the present invention, in combination and/or alternation with one or
more other effective antiproliferative agent, such as an antineoplastic
agent, optionally in a pharmaceutically acceptable carrier, in the
manufacture of a medicament for the treatment and/or prophylaxis of an
abnormal cellular proliferation, such as tumors and cancer, in a host,
preferably a human.

[0101] The invention also provides synthetic methods useful for preparing
the compounds of the invention, as well as intermediates disclosed herein
that are useful in the preparation of the compounds of the present
invention.

[0102] The invention as disclosed herein is method and composition for the
treatment of HIV, hepatitis B or C, or abnormal cellular proliferation,
in humans or other host animals, that includes administering a
therapeutically effective amount of a β-D- or β-L-nucleoside
derivatives, a pharmaceutically acceptable derivative, including a
compound which has been alkylated or acylated on sugar or phosphonate
moiety, or on the purine or pyrimidine, or a pharmaceutically acceptable
salt thereof, optionally in a pharmaceutically acceptable carrier. The
compounds of this invention either possess antiviral (i.e., anti-HIV-1,
anti-HIV-2, anti-hepatitis B/C virus) activity or antiproliferative
activity, or are metabolized to a compound that exhibits such activity.
The invention as disclosed herein also includes the process for the
preparation of such β-D- or β-L-nucleoside derivatives.

Stereoisomerism and Polymorphism

[0103] The compounds of the present invention may have asymmetric centers
and occur as racemates, racemic mixtures, individual diastereomers or
enantiomers, with all isomeric forms being included in the present
invention. Compounds of the present invention having a chiral center may
exist in and be isolated in optically active and racemic forms. Some
compounds may exhibit polymorphism. The present invention encompasses
racemic, optically-active, polymorphic, or stereoisomeric form, or
mixtures thereof, of a compound of the invention, which possess the
useful properties described herein. Optically active forms of the
compounds can be prepared using any method known in the art, including
but not limited to by resolution of the racemic form by recrystallization
techniques, by synthesis from optically-active starting materials; by
chiral synthesis, or by chromatographic separation using a chiral
stationary phase or by enzymatic resolution.

DEFINITIONS

[0104] Listed below are definitions of various terms used to describe this
invention. These definitions apply to the terms as they are used
throughout this specification and claims, unless otherwise limited in
specific instances, either individually or as part of a larger group.

[0105] The term "aryl," as used herein, refers to a mono- or polycyclic
carbocyclic ring system containing at least one aromatic ring, including,
but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, and
indenyl.

[0106] The term "heteroaryl," as used herein, refers to a mono- or
polycyclic aromatic radical having one or more ring atom selected from S,
O and N; and the remaining ring atoms are carbons, wherein any N or S
contained within the ring may be optionally oxidized. Heteroaryl
includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl,
pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl,
thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,
isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl.

[0107] In accordance with the invention, any of the aryls, substituted
aryls, heteroaryls and substituted heteroaryls described herein, can be
any aromatic group. Aromatic groups can be substituted or unsubstituted.

[0109] The term "C2-C8 alkenyl," or "C2-C20 alkenyl,"
as used herein, refer to straight- or branched-chain hydrocarbon radicals
containing from two to eight carbon atoms having at least one
carbon-carbon double bond by the removal of a single hydrogen atom.
Alkenyl groups include, but are not limited to, for example, ethenyl,
propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl, and the
like.

[0110] The term "C2-C8 alkynyl," or "C2-C20 alkynyl,"
as used herein, refer to straight- or branched-chain hydrocarbon radicals
containing from two to eight carbon atoms having at least one
carbon-carbon triple bond by the removal of a single hydrogen atom.
Representative alkynyl groups include, but are not limited to, for
example, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octynyl, and the like.

[0111] The term "C3-C8-cycloalkyl", or
"C3-C12-cycloalkyl," as used herein, refers to a monocyclic or
polycyclic saturated carbocyclic ring compound. Examples of
C3-C8-cycloalkyl include, but not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl; and
examples of C3-C12-cycloalkyl include, but not limited to,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl,
and bicyclo[2.2.2]octyl.

[0112] The terms "C2-C8 alkylene," or "C2-C8
alkenylene," as used herein, refer to saturated or unsaturated
respectively, straight- or branched-chain hydrocarbon di-radicals
containing between two and eight carbon atoms, while the diradical may
reside at the same or different carbon atoms.

[0113] The term "C3-C8 cycloalkenyl," or "C3-C12
cycloalkenyl" as used herein, refers to monocyclic or polycyclic
carbocyclic ring compound having at least one carbon-carbon double bond.
Examples of C3-C8 cycloalkenyl include, but not limited to,
cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,
cyclooctenyl, and the like; and examples of C3-C12 cycloalkenyl
include, but not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl,
cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like.

[0114] It is understood that any alkyl, alkenyl, alkynyl and cycloalkyl
moiety described herein can also be an aliphatic group, an alicyclic
group or a heterocyclic group. An "aliphatic" group is a non-aromatic
moiety that may contain any combination of carbon atoms, hydrogen atoms,
halogen atoms, oxygen, nitrogen or other atoms, and optionally contain
one or more units of unsaturation, e.g., double and/or triple bonds. An
aliphatic group may be straight chained, branched or cyclic and
preferably contains between about 1 and about 24 carbon atoms, more
typically between about 1 and about 12 carbon atoms. In addition to
aliphatic hydrocarbon groups, aliphatic groups include, for example,
polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and
polyimines, for example. Such aliphatic groups may be further
substituted.

[0115] The term "alicyclic," as used herein, denotes a monovalent group
derived from a monocyclic or bicyclic saturated carbocyclic ring compound
by the removal of a single hydrogen atom. Examples include, but not
limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl. Such alicyclic groups may
be further substituted.

[0116] The terms "heterocyclic" or "heterocycloalkyl" can be used
interchangeably and referred to a non-aromatic ring or a bi- or
tri-cyclic group fused system, where (i) each ring system contains at
least one heteroatom independently selected from oxygen, sulfur and
nitrogen, (ii) each ring system can be saturated or unsaturated (iii) the
nitrogen and sulfur heteroatoms may optionally be oxidized, (iv) the
nitrogen heteroatom may optionally be quaternized, (v) any of the above
rings may be fused to an aromatic ring, and (vi) the remaining ring atoms
are carbon atoms which may be optionally oxo-substituted. Representative
heterocyclic groups include, but are not limited to, 1,3-dioxolane,
pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,
piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl,
thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, and
tetrahydrofuryl. Such heterocyclic groups may be further substituted.

[0118] The term "halogen," as used herein, refers to an atom selected from
fluorine, chlorine, bromine and iodine.

[0119] The term "hydroxy activating group", as used herein, refers to a
labile chemical moiety which is known in the art to activate a hydroxyl
group so that it will depart during synthetic procedures such as in a
substitution or in an elimination reactions. Examples of hydroxyl
activating group include, but not limited to, mesylate, tosylate,
triflate, p-nitrobenzoate, phosphonate and the like.

[0120] The term "activated hydroxy", as used herein, refers to a hydroxy
group activated with a hydroxyl activating group, as defined above,
including mesylate, tosylate, triflate, p-nitrobenzoate, phosphonate
groups, for example.

[0121] The term "hydroxy protecting group" or "thiol protecting group," as
used herein, refers to a labile chemical moiety which is known in the art
to protect a hydroxyl group or thiol against undesired reactions during
synthetic procedures. After said synthetic procedure(s) the hydroxy
protecting group as described herein may be selectively removed. Hydroxy
protecting groups as known in the art are described generally in T. H.
Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd
edition, John Wiley & Sons, New York (1999). Examples of hydroxyl
protecting groups include benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl,
4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, methoxycarbonyl,
tert-butoxycarbonyl, isopropoxycarbonyl, diphenylmethoxycarbonyl,
2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl,
2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl,
trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, t-butyl,
2,2,2-trichloroethyl, 2-trimethylsilyl ethyl, 1,1-dimethyl-2-propenyl,
3-methyl-3-butenyl, allyl, benzyl, para-methoxybenzyldiphenylmethyl,
triphenylmethyl (trityl), tetrahydrofuryl, methoxymethyl,
methylthiomethyl, benzyloxymethyl, 2,2,2-triehloroethoxymethyl,
2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, para-toluenesulfonyl,
trimethylsilyl, triethylsilyl, triisopropylsilyl, and the like. Preferred
hydroxyl protecting groups for the present invention are acetyl (Ac or
--C(O)CH3), benzoyl (Bz or --C(O)C6H5), and trimethylsilyl
(TMS or --Si(CH3)3).

[0122] The term "protected hydroxy," as used herein, refers to a hydroxy
group protected with a hydroxy protecting group, as defined above,
including benzoyl, acetyl, trimethylsilyl, triethylsilyl, methoxymethyl
groups, for example.

[0123] The term "hydroxy prodrug group", as used herein, refers to a
promoiety group which is known in the art to change the physicochemical,
and hence the biological properties of a parent drug in a transient
manner by covering or masking the hydroxy group. After said synthetic
procedure(s), the hydroxy prodrug group as described herein must be
capable of reverting back to hydroxy group in vivo. Hydroxy prodrug
groups as known in the art are described generally in Kenneth B. Sloan,
Prodrugs, Topical and Ocular Drug Delivery, (Drugs and the Pharmaceutical
Sciences; Volume 53), Marcel Dekker, Inc., New York (1992).

[0124] The term "amino protecting group," as used herein, refers to a
labile chemical moiety which is known in the art to protect an amino
group against undesired reactions during synthetic procedures. After said
synthetic procedure(s) the amino protecting group as described herein may
be selectively removed. Amino protecting groups as known in the art are
described generally in T. H. Greene and P. G. M. Wuts, Protective Groups
in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).
Examples of amino protecting groups include, but are not limited to,
t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, and the
like.

[0125] The term "leaving group" means a functional group or atom which can
be displaced by another functional group or atom in a substitution
reaction, such as a nucleophilic substitution reaction. By way of
example, representative leaving groups include chloro, bromo and iodo
groups; sulfonic ester groups, such as mesylate, tosylate, brosylate,
nosylate and the like; and acyloxy groups, such as acetoxy,
trifluoroacetoxy and the like.

[0126] The term "protected amino," as used herein, refers to an amino
group protected with an amino protecting group as defined above.

[0127] The term "aprotic solvent," as used herein, refers to a solvent
that is relatively inert to proton activity, i.e., not acting as a
proton-donor. Examples include, but are not limited to, hydrocarbons,
such as hexane and toluene, for example, halogenated hydrocarbons, such
as, for example, methylene chloride, ethylene chloride, chloroform, and
the like, heterocyclic compounds, such as, for example, tetrahydrofuran
and N-methylpyrrolidinone, and ethers such as diethyl ether,
bis-methoxymethyl ether. Such compounds are well known to those skilled
in the art, and it will be obvious to those skilled in the art that
individual solvents or mixtures thereof may be preferred for specific
compounds and reaction conditions, depending upon such factors as the
solubility of reagents, reactivity of reagents and preferred temperature
ranges, for example. Further discussions of aprotic solvents may be found
in organic chemistry textbooks or in specialized monographs, for example:
Organic Solvents Physical Properties and Methods of Purification, 4th
ed., edited by John A. Riddick et al., Vol. II, in the Techniques of
Chemistry Series, John Wiley & Sons, NY, 1986.

[0128] The term "protic solvent` as used herein, refers to a solvent that
tends to provide protons, such as an alcohol, for example, methanol,
ethanol, propanol, isopropanol, butanol, t-butanol, and the like. Such
solvents are well known to those skilled in the art, and it will be
obvious to those skilled in the art that individual solvents or mixtures
thereof may be preferred for specific compounds and reaction conditions,
depending upon such factors as the solubility of reagents, reactivity of
reagents and preferred temperature ranges, for example. Further
discussions of protogenic solvents may be found in organic chemistry
textbooks or in specialized monographs, for example: Organic Solvents
Physical Properties and Methods of Purification, 4th ed., edited by John
A. Riddick et al., Vol. II, in the Techniques of Chemistry Series, John
Wiley & Sons, NY, 1986.

[0129] The term "hydrogen" includes deuterium. In general, the
identification of an element embraces the isotopes of the element, as
suitable for the preparation a pharmaceutical.

[0130] Combinations of substituents and variables envisioned by this
invention are only those that result in the formation of stable
compounds. The term "stable", as used herein, refers to compounds which
possess stability sufficient to allow manufacture and which maintains the
integrity of the compound for a sufficient period of time to be useful
for the purposes detailed herein (e.g., therapeutic or prophylactic
administration to a subject).

[0131] The synthesized compounds can be separated from a reaction mixture
and further purified by a method such as column chromatography, high
pressure liquid chromatography, or recrystallization. As can be
appreciated by the skilled artisan, further methods of synthesizing the
compounds of the Formula herein will be evident to those of ordinary
skill in the art. Additionally, the various synthetic steps may be
performed in an alternate sequence or order to give the desired
compounds. Synthetic chemistry transformations and protecting group
methodologies (protection and deprotection) useful in synthesizing the
compounds described herein are known in the art and include, for example,
those such as described in R. Larock, Comprehensive Organic
Transformations, 2nd Ed. Wiley-VCH (1999); T. W. Greene and P. G. M.
Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley and
Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for
Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,
Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons
(1995), and subsequent editions thereof.

[0132] The term "subject" as used herein refers to an animal. Preferably
the animal is a mammal. More preferably the mammal is a human. A subject
also refers to, for example, dogs, cats, horses, cows, pigs, guinea pigs,
fish, birds and the like.

[0133] The compounds of this invention may be modified by appending
appropriate functionalities to enhance selective biological properties.
Such modifications are known in the art and may include those which
increase biological penetration into a given biological system (e.g.,
blood, lymphatic system, central nervous system), increase oral
availability, increase solubility to allow administration by injection,
alter metabolism and alter rate of excretion.

[0134] The compounds described herein contain one or more asymmetric
centers and thus give rise to enantiomers, diastereomers, and other
stereoisomeric forms that may be defined, in terms of absolute
stereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids. The
present invention is meant to include all such possible isomers, as well
as their racemic and optically pure forms. Optical isomers may be
prepared from their respective optically active precursors by the
procedures described above, or by resolving the racemic mixtures. The
resolution can be carried out in the presence of a resolving agent, by
chromatography or by repeated crystallization or by some combination of
these techniques which are known to those skilled in the art. Further
details regarding resolutions can be found in Jacques, et al.,
Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). When
the compounds described herein contain olefinic double bonds, other
unsaturation, or other centers of geometric asymmetry, and unless
specified otherwise, it is intended that the compounds include both E and
Z geometric isomers, or cis- and trans- isomers. Likewise, all tautomeric
forms are also intended to be included. Tautomers may be in cyclic or
acyclic. The configuration of any carbon-carbon double bond appearing
herein is selected for convenience only and is not intended to designate
a particular configuration unless the text so states; thus a
carbon-carbon double bond or carbon-heteroatom double bond depicted
arbitrarily herein as trans may be cis, trans, or a mixture of the two in
any proportion.

[0135] As used herein, the term "pharmaceutically acceptable salt" refers
to those salts which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower animals
without undue toxicity, irritation, allergic response and the like, and
are commensurate with a reasonable benefit/risk ratio. Pharmaceutically
acceptable salts are well known in the art. For example, S. M. Berge, et
al. describes pharmaceutically acceptable salts in detail in J.
Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in
situ during the final isolation and purification of the compounds of the
invention, or separately by reacting the free base function with a
suitable organic acid. Examples of pharmaceutically acceptable salts
include, but are not limited to, nontoxic acid addition salts are salts
of an amino group formed with inorganic acids such as hydrochloric acid,
hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or
with organic acids such as acetic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other methods used
in the art such as ion exchange. Other pharmaceutically acceptable salts
include, but are not limited to, adipate, alginate, ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,
lauryl sulfate, malate, maleate, malonate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,
pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,
pivalate, propionate, stearate, succinate, sulfate, tartrate,
thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the
like. Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate, nontoxic
ammonium, quaternary ammonium, and amine cations formed using counterions
such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate,
alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.

[0136] As used herein, the term "pharmaceutically acceptable ester" refers
to esters which hydrolyze in vivo and include those that break down
readily in the human body to leave the parent compound or a salt thereof.
Suitable ester groups include, for example, those derived from
pharmaceutically acceptable aliphatic carboxylic acids, particularly
alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each
alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.
Examples of particular esters include, but are not limited to, formates,
acetates, propionates, butyrates, acrylates and ethylsuccinates.

[0137] The term "pharmaceutically acceptable prodrugs" as used herein
refers to those prodrugs of the compounds of the present invention which
are, within the scope of sound medical judgment, suitable for use in
contact with the tissues of humans and lower animals with undue toxicity,
irritation, allergic response, and the like, commensurate with a
reasonable benefit/risk ratio, and effective for their intended use, as
well as the zwitterionic forms, where possible, of the compounds of the
present invention. "Prodrug", as used herein means a compound which is
convertible in vivo by metabolic means (e.g. by hydrolysis) to a compound
of the invention. Various forms of prodrugs are known in the art, for
example, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier
(1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, Academic
Press (1985); Krogsgaard-Larsen, et al., (ed.). "Design and Application
of Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191
(1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1-38
(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988);
Higuchi and Stella (eds.) Prodrugs as Novel Drug Delivery Systems,
American Chemical Society (1975); Bernard Testa & Joachim Mayer,
"Hydrolysis In Drug And Prodrug Metabolism: Chemistry, Biochemistry And
Enzymology," John Wiley and Sons, Ltd. (2002); and J. Rautio et al,
"Prodrugs: design and clinical applications", Nature Review--Drug
Discovery, 7, 255-270 (2008).

[0138] The present invention also relates to solvates of the compounds of
Formula (I), for example hydrates.

[0139] It is to be understood that when a variable appears two or more
times in a formula set forth herein, each occurrence of the variable is
independently selected from the identities set forth for that variable.

[0140] This invention also encompasses pharmaceutical compositions
containing, and methods of treating viral infections through
administering, pharmaceutically acceptable prodrugs of compounds of the
invention. For example, compounds of the invention having free amino,
amido, hydroxy or carboxylic groups can be converted into prodrugs.
Prodrugs include compounds wherein an amino acid residue, or a
polypeptide chain of two or more (e.g., two, three or four) amino acid
residues is covalently joined through an amide or ester bond to a free
amino, hydroxy or carboxylic acid group of compounds of the invention.
The amino acid residues include but are not limited to the 20 naturally
occurring amino acids commonly designated by three letter symbols and
also includes 4-hydroxyproline, hydroxylysine, demosine, isodemosine,
3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,
citrulline, homocysteine, homoserine, ornithine and methionine sulfone.
Additional types of prodrugs are also encompassed. For instance, free
carboxyl groups can be derivatized as amides or alkyl esters. Free
hydroxy groups may be derivatized using groups including but not limited
to hemisuccinates, phosphate esters, dimethylaminoacetates, and
phosphoryloxymethyloxycarbonyls, as outlined in Advanced Drug Delivery
Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxy and amino groups
are also included, as are carbonate prodrugs, sulfonate esters and
sulfate esters of hydroxy groups. Derivatization of hydroxy groups as
(acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may be
an alkyl ester, optionally substituted with groups including but not
limited to ether, amine and carboxylic acid functionalities, or where the
acyl group is an amino acid ester as described above, are also
encompassed. Prodrugs of this type are described in J. Med. Chem. 1996,
39, 10. Free amines can also be derivatized as amides, sulfonamides or
phosphonamides. All of these prodrug moieties may incorporate groups
including but not limited to ether, amine and carboxylic acid
functionalities.

Pharmaceutical Compositions.

[0141] The pharmaceutical compositions of the present invention comprise a
therapeutically effective amount of a compound of the present invention
formulated together with one or more pharmaceutically acceptable carriers
or excipients.

[0142] As used herein, the term "pharmaceutically acceptable carrier or
excipient" means a non-toxic, inert solid, semi-solid or liquid filler,
diluent, encapsulating material or formulation auxiliary of any type.
Some examples of materials which can serve as pharmaceutically acceptable
carriers are sugars such as lactose, glucose and sucrose; starches such
as corn starch and potato starch; cellulose and its derivatives such as
sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil, safflower
oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as
propylene glycol; esters such as ethyl oleate and ethyl laurate; agar;
buffering agents such as magnesium hydroxide and aluminun hydroxide;
alginic acid; pyrogen-free water; isotonic saline; Ringer's solution;
ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating agents,
sweetening, flavoring and perfuming agents, preservatives and
antioxidants can also be present in the composition, according to the
judgment of the formulator.

[0143] The pharmaceutical compositions of this invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted reservoir,
preferably by oral administration or administration by injection. The
pharmaceutical compositions of this invention may contain any
conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or
vehicles. In some cases, the pH of the formulation may be adjusted with
pharmaceutically acceptable acids, bases or buffers to enhance the
stability of the formulated compound or its delivery form. The term
parenteral as used herein includes subcutaneous, intracutaneous,
intravenous, intramuscular, intraarticular, intraarterial, intrasynovial,
intrasternal, intrathecal, intralesional and intracranial injection or
infusion techniques.

[0145] Injectable preparations, for example, sterile injectable aqueous or
oleaginous suspensions, may be formulated according to the known art
using suitable dispersing or wetting agents and suspending agents. The
sterile injectable preparation may also be a sterile injectable solution,
suspension or emulsion in a nontoxic parenterally acceptable diluent or
solvent, for example, as a solution in 1,3-butanediol. Among the
acceptable vehicles and solvents that may be employed are water, Ringer's
solution, U.S.P. and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil can be employed
including synthetic mono- or diglycerides. In addition, fatty acids such
as oleic acid are used in the preparation of injectables.

[0146] The injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which can be
dissolved or dispersed in sterile water or other sterile injectable
medium prior to use.

[0147] In order to prolong the effect of a drug, it is often desirable to
slow the absorption of the drug from subcutaneous or intramuscular
injection. This may be accomplished by the use of a liquid suspension of
crystalline or amorphous material with poor water solubility. The rate of
absorption of the drug then depends upon its rate of dissolution, which,
in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally administered drug
form is accomplished by dissolving or suspending the drug in an oil
vehicle. Injectable depot forms are made by forming microencapsule
matrices of the drug in biodegradable polymers such as
polylactide-polyglycolide. Depending upon the ratio of drug to polymer
and the nature of the particular polymer employed, the rate of drug
release can be controlled. Examples of other biodegradable polymers
include poly(orthoesters) and poly(anhydrides). Depot injectable
formulations are also prepared by entrapping the drug in liposomes or
microemulsions that are compatible with body tissues.

[0148] Compositions for rectal or vaginal administration are preferably
suppositories which can be prepared by mixing the compounds of this
invention with suitable non-irritating excipients or carriers such as
cocoa butter, polyethylene glycol or a suppository wax which are solid at
ambient temperature but liquid at body temperature and therefore melt in
the rectum or vaginal cavity and release the active compound.

[0149] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms, the
active compound is mixed with at least one inert, pharmaceutically
acceptable excipient or carrier such as sodium citrate or dicalcium
phosphate and/or: a) fillers or extenders such as starches, lactose,
sucrose, glucose, mannitol, and silicic acid, b) binders such as, for
example, carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as
glycerol, d) disintegrating agents such as agar-agar, calcium carbonate,
potato or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, e) solution retarding agents such as paraffin, f) absorption
accelerators such as quaternary ammonium compounds, g) wetting agents
such as, for example, cetyl alcohol and glycerol monostearate, h)
absorbents such as kaolin and bentonite clay, and i) lubricants such as
talc, calcium stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl sulfate, and mixtures thereof. In the case of capsules,
tablets and pills, the dosage form may also comprise buffering agents.

[0150] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such excipients as
lactose or milk sugar as well as high molecular weight polyethylene
glycols and the like.

[0151] The solid dosage forms of tablets, dragees, capsules, pills, and
granules can be prepared with coatings and shells such as enteric
coatings and other coatings well known in the pharmaceutical formulating
art. They may optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract, optionally, in
a delayed manner. Examples of embedding compositions that can be used
include polymeric substances and waxes.

[0152] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams, lotions,
gels, powders, solutions, sprays, inhalants or patches. The active
component is admixed under sterile conditions with a pharmaceutically
acceptable carrier and any needed preservatives or buffers as may be
required. Ophthalmic formulation, ear drops, eye ointments, powders and
solutions are also contemplated as being within the scope of this
invention.

[0154] Powders and sprays can contain, in addition to the compounds of
this invention, excipients such as lactose, talc, silicic acid, aluminum
hydroxide, calcium silicates and polyamide powder, or mixtures of these
substances. Sprays can additionally contain customary propellants such as
chlorofluorohydrocarbons.

[0155] Transdermal patches have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms can be
made by dissolving or dispensing the compound in the proper medium.
Absorption enhancers can also be used to increase the flux of the
compound across the skin. The rate can be controlled by either providing
a rate controlling membrane or by dispersing the compound in a polymer
matrix or gel.

[0156] For pulmonary delivery, a therapeutic composition of the invention
is formulated and administered to the patient in solid or liquid
particulate form by direct administration e.g., inhalation into the
respiratory system. Solid or liquid particulate forms of the active
compound prepared for practicing the present invention include particles
of respirable size: that is, particles of a size sufficiently small to
pass through the mouth and larynx upon inhalation and into the bronchi
and alveoli of the lungs. Delivery of aerosolized therapeutics,
particularly aerosolized antibiotics, is known in the art (see, for
example U.S. Pat. No. 5,767,068 to VanDevanter et al., U.S. Pat. No.
5,508,269 to Smith et al., and WO 98/43,650 by Montgomery, all of which
are incorporated herein by reference). A discussion of pulmonary delivery
of antibiotics is also found in U.S. Pat. No. 6,014,969, incorporated
herein by reference.

[0157] According to the methods of treatment of the present invention,
viral infections, conditions are treated or prevented in a patient such
as a human or another animal by administering to the patient a
therapeutically effective amount of a compound of the invention, in such
amounts and for such time as is necessary to achieve the desired result.

[0158] By a "therapeutically effective amount" of a compound of the
invention is meant an amount of the compound which confers a therapeutic
effect on the treated subject, at a reasonable benefit/risk ratio
applicable to any medical treatment. The therapeutic effect may be
objective (i.e., measurable by some test or marker) or subjective (i.e.,
subject gives an indication of or feels an effect). A therapeutically
effective amount of the compound described above may range from about 0.1
mg/Kg to about 500 mg/Kg, preferably from about 1 to about 50 mg/Kg.
Effective doses will also vary depending on route of administration, as
well as the possibility of co-usage with other agents. It will be
understood, however, that the total daily usage of the compounds and
compositions of the present invention will be decided by the attending
physician within the scope of sound medical judgment. The specific
therapeutically effective dose level for any particular patient will
depend upon a variety of factors including the disorder being treated and
the severity of the disorder; the activity of the specific compound
employed; the specific composition employed; the age, body weight,
general health, sex and diet of the patient; the time of administration,
route of administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination or
contemporaneously with the specific compound employed; and like factors
well known in the medical arts.

[0159] The total daily dose of the compounds of this invention
administered to a human or other animal in single or in divided doses can
be in amounts, for example, from 0.01 to 50 mg/kg body weight or more
usually from 0.1 to 25 mg/kg body weight. Single dose compositions may
contain such amounts or submultiples thereof to make up the daily dose.
In general, treatment regimens according to the present invention
comprise administration to a patient in need of such treatment from about
10 mg to about 1000 mg of the compound(s) of this invention per day in
single or multiple doses.

[0160] The compounds of the invention described herein can, for example,
be administered by injection, intravenously, intraarterially,
subdermally, intraperitoneally, intramuscularly, or subcutaneously; or
orally, buccally, nasally, transmucosally, topically, in an ophthalmic
preparation, or by inhalation, with a dosage ranging from about 0.1 to
about 500 mg/kg of body weight, alternatively dosages between 1 mg and
1000 mg/dose, every 4 to 120 hours, or according to the requirements of
the particular drug. The methods herein contemplate administration of a
therapeutically effective amount of compound or compound composition to
achieve the desired or stated effect. Typically, the pharmaceutical
compositions of this invention will be administered from about 1 to about
6 times per day or alternatively, as a continuous infusion. Such
administration can be used as a chronic or acute therapy. The amount of
active ingredient that may be combined with pharmaceutically exipients or
carriers to produce a single dosage form will vary depending upon the
host treated and the particular mode of administration. A typical
preparation will contain from about 5% to about 95% active compound
(w/w). Alternatively, such preparations may contain from about 20% to
about 80% active compound.

[0161] Lower or higher doses than those recited above may be required.
Specific dosage and treatment regimens for any particular patient will
depend upon a variety of factors, including the activity of the specific
compound employed, the age, body weight, general health status, sex,
diet, time of administration, rate of excretion, drug combination, the
severity and course of the disease, condition or symptoms, the patient's
disposition to the disease, condition or symptoms, and the judgment of
the treating physician.

[0162] Upon improvement of a patient's condition, a maintenance dose of a
compound, composition or combination of this invention may be
administered, if necessary. Subsequently, the dosage or frequency of
administration, or both, may be reduced, as a function of the symptoms,
to a level at which the improved condition is retained when the symptoms
have been alleviated to the desired level. Patients may, however, require
intermittent treatment on a long-term basis upon any recurrence of
disease symptoms.

[0163] When the compositions of this invention comprise a combination of a
compound of the invention described herein and one or more additional
therapeutic or prophylactic agents, both the compound and the additional
agent should be present at dosage levels of between about 1 to 100%, and
more preferably between about 5 to 95% of the dosage normally
administered in a monotherapy regimen. The additional agents may be
administered separately, as part of a multiple dose regimen, from the
compounds of this invention. Alternatively, those agents may be part of a
single dosage form, mixed together with the compounds of this invention
in a single composition.

[0164] The said "additional therapeutic or prophylactic agents" includes
but not limited to, immune therapies (eg. interferon), therapeutic
vaccines, antifibrotic agents, anti-inflammatory agents such as
corticosteroids or NSAIDs, bronchodilators such as beta-2 adrenergic
agonists and xanthines (e.g. theophylline), mucolytic agents,
anti-muscarinics, anti-leukotrienes, inhibitors of cell adhesion (e.g.
ICAM antagonists), anti-oxidants (eg N-acetylcysteine), cytokine
agonists, cytokine antagonists, lung surfactants and/or antimicrobial and
anti-viral agents (eg ribavirin and amantidine). The compositions
according to the invention may also be used in combination with gene
replacement therapy.

[0165] Unless otherwise defined, all technical and scientific terms used
herein are accorded the meaning commonly known to one of ordinary skill
in the art. All publications, patents, published patent applications, and
other references mentioned herein are hereby incorporated by reference in
their entirety.

Pharmaceutically Acceptable Derivatives

[0166] The compound of the present invention can be administered as any
derivative that upon administration to the recipient is capable of
providing directly or indirectly, the parent compound. Further, the
modifications can affect the biological activity of the compound, in some
cases increasing the activity over the parent compound. This can easily
be assessed by preparing the derivative and testing its antiviral and
anti-proliferative activity according to the methods described herein, or
other method known to those skilled in the art.

[0167] In cases where compounds are suficiently basic or acidic to form
stable nontoxic acid or base salts, administration of the compound as a
pharmaceutically acceptable salt may be appropriate. Examples of
pharmaceutically acceptable salts are organic acid addition salts formed
with acids, which form a physiological acceptable anion, for example,
tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,
succinate, benzoate, ascorbate, α-ketoglutarate and
α-glycerophosphate. Suitable inorganic salts may also be formed,
including sulfate, nitrate, bicarbonate, and carbonate salts.

[0168] Pharmaceutically acceptable salts may be obtained using standard
procedures well known in the art, for example by reacting a sufficiently
basic compound such as an amine with a suitable acid affording a
physiologically acceptable anion. Alkali metal (for example, sodium,
potassium or lithium) or alkaline earth metal (for example calcium) salts
of carboxylic acids can also be made.

[0169] Any of the nucleosides described herein can be administrated as a
nucleotide prodrug to increase the activity, bioavailability, stability
or otherwise alter the properties of the nucleoside. A number of
nucleotide prodrug ligands are known. In general, alkylation, acylation
or other lipophilic modification of the mono, di or triphosphate of the
nucleoside will increase the stability of the nucleotide. Examples of
substituent groups that can replace one or more hydrogens on the
phosphate moiety are alkyl, aryl, steroids, carbohydrates, including
sugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jones
and N. Bischofberger, Antiviral Research, 27 (1995) 1-17. Any of these
can be used in combination with the disclosed nucleosides to achieve a
desired effect.

[0173] Similarly, the 5'-phosphonate can also be provided as various
phosphonate prodrug to increase the activity, bioavailability, stability
or otherwise alter the properties of the phosphonate. A number of
phosphonate prodrug ligands are known. In general, alkylation, acylation
or other lipophilic modification of one or more hydroxy on the
phosphonate moiety can be used to achieve a desired effect.

Combination and Alternation Therapy for HIV, HBV or HCV

[0174] It has been recognized that drug-resistant variants of HIV, HBV and
HCV can emerge after prolonged treatment with an antiviral agent. Drug
resistance most typically occurs by mutation of a gene that encodes for a
protein such as an enzyme used in viral replication, and most typically
in the case of HIV, reverse transcriptase, protease, or DNA polymerase,
and in the case of HBV, DNA polymerase, or in the case of HCV, RNA
polymerase, protease, or helicase. Recently, it has been demonstrated
that the efficacy of a drug against HIV infection can be prolonged,
augmented, or restored by administering the compound in combination or
alternation with a second, and perhaps third, antiviral compound that
induces a different mutation from that caused by the principle drug.
Alternatively, the pharmacokinetics, biodistribution, or other parameter
of the drug can be altered by such combination or alternation therapy. In
general, combination therapy is typically preferred over alternation
therapy because it induces multiple simultaneous stresses on the virus.

[0175] The second antiviral agent for the treatment of HIV, in one
embodiment, can be a reverse transcriptase inhibitor (a "RTI"), which can
be either a synthetic nucleoside (a "NRTI") or a non-nucleoside compound
(a "NNRTI"). In an alternative embodiment, in the case of HIV, the second
(or third) antiviral agent can be a protease inhibitor. In other
embodiments, the second (or third) compound can be a pyrophosphate
analog, or a fusion binding inhibitor. A list compiling resistance data
collected in vitro and in vivo for a number of antiviral compounds is
found in Schinazi, et al, Mutations in retroviral genes associated with
drug resistance, International Antiviral News, 1997.

[0182] The active compound can also be administered in combination or
alternation with ribavarin, interferon, interleukin or a stabilized
prodrug of any of them. More broadly described, the compound can be
administered in combination or alternation with any of the anti-HCV drugs
listed below.

[0183] In another embodiment, the compounds, when used as an
antiproliferative, can be administered in combination with another
compound that increases the effectiveness of the therapy, including but
not limited to an antifolate, a 5-fluoropyrimidine (including
5-fluorouracil), a cytidine analogue such as β-L-1,3-dioxolanyl
cytidine or β-L-1,3-dioxolanyl 5-fluorocytidine, antimetabolites
(including purine antimetabolites, cytarabine, fudarabine, floxuridine,
6-mercaptopurine, methotrexate, and 6-thioguanine), hydroxyurea, mitotic
inhibitors (including CPT-11, Etoposide (VP-21), taxol, and vinca
alkaloids such as vincristine and vinblastine, an alkylating agent
(including but not limited to busulfan, chlorambucil, cyclophosphamide,
ifofamide, mechlorethamine, melphalan, and thiotepa), nonclassical
alkylating agents, platinum containing compounds, bleomycin, an
anti-tumor antibiotic, an anthracycline such as doxorubicin and
dannomycin, an anthracenedione, topoisomerase II inhibitors, hormonal
agents (including but not limited to corticosteroids (dexamethasone,
prednisone, and methylprednisone), androgens such as fluoxymesterone and
methyltestosterone, estrogens such as diethylstilbesterol, antiestrogens
such as tamoxifen, LHRH analogues such as leuprolide, antiandrogens such
as flutamide, aminoglutethimide, megestrol acetate, and
medroxyprogesterone), asparaginase, carmustine, lomustine,
hexamethyl-melamine, dacarbazine, mitotane, streptozocin, cisplatin,
carboplatin, levamasole, and leucovorin. The compounds of the present
invention can also be used in combination with enzyme therapy agents and
immune system modulators such as an interferon, interleukin, tumor
necrosis factor, macrophage colony-stimulating factor and colony
stimulating factor.

[0184] Although the invention has been described with respect to various
preferred embodiments, it is not intended to be limited thereto, but
rather those skilled in the art will recognize that variations and
modifications may be made therein which are within the spirit of the
invention and the scope of the appended claims.

Antiviral Activity

[0185] An inhibitory amount or dose of the compounds of the present
invention may range from about 0.01 mg/Kg to about 500 mg/Kg,
alternatively from about 1 to about 100 mg/Kg. Inhibitory amounts or
doses will also vary depending on route of administration, as well as the
possibility of co-usage with other agents.

[0186] According to the methods of treatment of the present invention,
viral infections are treated or prevented in a subject such as a human or
lower mammal by administering to the subject an anti-hepatitis C virally
effective amount or an inhibitory amount of a compound of the present
invention, in such amounts and for such time as is necessary to achieve
the desired result. An additional method of the present invention is the
treatment of biological samples with an inhibitory amount of a compound
of composition of the present invention in such amounts and for such time
as is necessary to achieve the desired result.

[0187] The term "anti-hepatitis C virally effective amount" of a compound
of the invention, as used herein, mean a sufficient amount of the
compound so as to decrease the viral load in a biological sample or in a
subject (e.g., resulting in at least 10%, preferably at least 50%, more
preferably at least 80%, and most preferably at least 90% or 95%,
reduction in viral load). As well understood in the medical arts, an
anti-hepatitis C virally effective amount of a compound of this invention
will be at a reasonable benefit/risk ratio applicable to any medical
treatment.

[0188] The term "inhibitory amount" of a compound of the present invention
means a sufficient amount to decrease the hepatitis C viral load in a
biological sample or a subject (e.g., resulting in at least 10%,
preferably at least 50%, more preferably at least 80%, and most
preferably at least 90% or 95%, reduction in viral load). It is
understood that when said inhibitory amount of a compound of the present
invention is administered to a subject it will be at a reasonable
benefit/risk ratio applicable to any medical treatment as determined by a
physician. The term "biological sample(s)," as used herein, means a
substance of biological origin intended for administration to a subject.
Examples of biological samples include, but are not limited to, blood and
components thereof such as plasma, platelets, subpopulations of blood
cells and the like; organs such as kidney, liver, heart, lung, and the
like; sperm and ova; bone marrow and components thereof; or stem cells.
Thus, another embodiment of the present invention is a method of treating
a biological sample by contacting said biological sample with an
inhibitory amount of a compound or pharmaceutical composition of the
present invention.

[0189] Upon improvement of a subject's condition, a maintenance dose of a
compound, composition or combination of this invention may be
administered, if necessary. Subsequently, the dosage or frequency of
administration, or both, may be reduced, as a function of the symptoms,
to a level at which the improved condition is retained when the symptoms
have been alleviated to the desired level, treatment should cease. The
subject may, however, require intermittent treatment on a long-term basis
upon any recurrence of disease symptoms.

[0190] It will be understood, however, that the total daily usage of the
compounds and compositions of the present invention will be decided by
the attending physician within the scope of sound medical judgment. The
specific inhibitory dose for any particular patient will depend upon a
variety of factors including the disorder being treated and the severity
of the disorder; the activity of the specific compound employed; the
specific composition employed; the age, body weight, general health, sex
and diet of the patient; the time of administration, route of
administration, and rate of excretion of the specific compound employed;
the duration of the treatment; drugs used in combination or coincidental
with the specific compound employed; and like factors well known in the
medical arts.

[0191] The total daily inhibitory dose of the compounds of this invention
administered to a subject in single or in divided doses can be in
amounts, for example, from 0.01 to 50 mg/kg body weight or more usually
from 0.1 to 25 mg/kg body weight. Single dose compositions may contain
such amounts or submultiples thereof to make up the daily dose. In
general, treatment regimens according to the present invention comprise
administration to a patient in need of such treatment from about 10 mg to
about 1000 mg of the compound(s) of this invention per day in single or
multiple doses.

[0192] Unless otherwise defined, all technical and scientific terms used
herein are accorded the meaning commonly known to one with ordinary skill
in the art. All publications, patents, published patent applications, and
other references mentioned herein are hereby incorporated by reference in
their entirety.

Antiviral Activity

[0193] An inhibitory amount or dose of the compounds of the present
invention may range from about 0.01 mg/Kg to about 500 mg/Kg,
alternatively from about 1 to about 100 mg/Kg. Inhibitory amounts or
doses will also vary depending on route of administration, as well as the
possibility of co-usage with other agents.

[0194] According to the methods of treatment of the present invention,
viral infections are treated or prevented in a subject such as a human or
lower mammal by administering to the subject an anti-hepatitis C virally
effective amount or an inhibitory amount of a compound of the present
invention, in such amounts and for such time as is necessary to achieve
the desired result. An additional method of the present invention is the
treatment of biological samples with an inhibitory amount of a compound
of composition of the present invention in such amounts and for such time
as is necessary to achieve the desired result.

[0195] The term "anti-hepatitis C virally effective amount" of a compound
of the invention, as used herein, mean a sufficient amount of the
compound so as to decrease the viral load in a biological sample or in a
subject (e.g., resulting in at least 10%, preferably at least 50%, more
preferably at least 80%, and most preferably at least 90% or 95%,
reduction in viral load). As well understood in the medical arts, an
anti-hepatitis C virally effective amount of a compound of this invention
will be at a reasonable benefit/risk ratio applicable to any medical
treatment.

[0196] The term "inhibitory amount" of a compound of the present invention
means a sufficient amount to decrease the hepatitis C viral load in a
biological sample or a subject (e.g., resulting in at least 10%,
preferably at least 50%, more preferably at least 80%, and most
preferably at least 90% or 95%, reduction in viral load). It is
understood that when said inhibitory amount of a compound of the present
invention is administered to a subject it will be at a reasonable
benefit/risk ratio applicable to any medical treatment as determined by a
physician. The term "biological sample(s)," as used herein, means a
substance of biological origin intended for administration to a subject.
Examples of biological samples include, but are not limited to, blood and
components thereof such as plasma, platelets, subpopulations of blood
cells and the like; organs such as kidney, liver, heart, lung, and the
like; sperm and ova; bone marrow and components thereof; or stem cells.
Thus, another embodiment of the present invention is a method of treating
a biological sample by contacting said biological sample with an
inhibitory amount of a compound or pharmaceutical composition of the
present invention.

[0197] Upon improvement of a subject's condition, a maintenance dose of a
compound, composition or combination of this invention may be
administered, if necessary. Subsequently, the dosage or frequency of
administration, or both, may be reduced, as a function of the symptoms,
to a level at which the improved condition is retained when the symptoms
have been alleviated to the desired level, treatment should cease. The
subject may, however, require intermittent treatment on a long-term basis
upon any recurrence of disease symptoms.

[0198] It will be understood, however, that the total daily usage of the
compounds and compositions of the present invention will be decided by
the attending physician within the scope of sound medical judgment. The
specific inhibitory dose for any particular patient will depend upon a
variety of factors including the disorder being treated and the severity
of the disorder; the activity of the specific compound employed; the
specific composition employed; the age, body weight, general health, sex
and diet of the patient; the time of administration, route of
administration, and rate of excretion of the specific compound employed;
the duration of the treatment; drugs used in combination or coincidental
with the specific compound employed; and like factors well known in the
medical arts.

[0199] The total daily inhibitory dose of the compounds of this invention
administered to a subject in single or in divided doses can be in
amounts, for example, from 0.01 to 50 mg/kg body weight or more usually
from 0.1 to 25 mg/kg body weight. Single dose compositions may contain
such amounts or submultiples thereof to make up the daily dose. In
general, treatment regimens according to the present invention comprise
administration to a patient in need of such treatment from about 10 mg to
about 1000 mg of the compound(s) of this invention per day in single or
multiple doses.

[0200] Unless otherwise defined, all technical and scientific terms used
herein are accorded the meaning commonly known to one with ordinary skill
in the art. All publications, patents, published patent applications, and
other references mentioned herein are hereby incorporated by reference in
their entirety.

Abbreviations

[0201] Abbreviations which may be used in the descriptions of the scheme
and the examples that follow are: Ac for acetyl; AcOH for acetic acid;
AIBN for azobisisobutyronitrile; BINAP for
2,2'-bis(diphenylphosphino)-1,1'-binaphthyl; Boc2O for
di-tert-butyl-dicarbonate; Boc for t-butoxycarbonyl; Bpoc for
1-methyl-1-(4-biphenylyl)ethyl carbonyl; Bz for benzoyl; Bn for benzyl;
BocNHOH for tert-butyl N-hydroxycarbamate; t-BuOK for potassium
tert-butoxide; Bu3SnH for tributyltin hydride; BOP for
(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium Hexafluorophosphate;
Brine for sodium chloride solution in water; CDI for carbonyldiimidazole;
CH2Cl2 for dichloromethane; CH3 for methyl; CH3CN for
acetonitrile; Cs2CO3 for cesium carbonate; CuCl for copper (I)
chloride; CuI for copper (I) iodide; dba for dibenzylidene acetone; dppb
for diphenylphosphino butane; DBU for 1,8-diazabicyclo[5.4.0]undec-7-ene;
DCC for N,N'-dicyclohexylcarbodiimide; DEAD for diethylazodicarboxylate;
DIAD for diisopropyl azodicarboxylate; DIPEA or (i-Pr)2EtN for
N,N,-diisopropylethyl amine; Dess-Martin periodinane for
1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one; DMAP for
4-dimethylaminopyridine; DME for 1,2-dimethoxyethane; DMF for
N,N-dimethylformamide; DMSO for dimethyl sulfoxide; DMT for
di(p-methoxyphenyl)phenylmethyl or dimethoxytrityl; DPPA for
diphenylphosphoryl azide; EDC for
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide; EDC HCl for
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; EtOAc for
ethyl acetate; EtOH for ethanol; Et2O for diethyl ether; HATU for
O-(7-azabenzotriazol-1-yl)-N,N,N',N',-tetramethyluronium
Hexafluorophosphate; HCl for hydrogen chloride; HOBT for
1-hydroxybenzotriazole; K2CO3 for potassium carbonate; n-BuLi
for n-butyl lithium; i-BuLi for i-butyl lithium; t-BuLi for t-butyl
lithium; PhLi for phenyl lithium; LDA for lithium diisopropylamide; LiTMP
for lithium 2,2,6,6-tetramethylpiperidinate; MeOH for methanol; Mg for
magnesium; MOM for methoxymethyl; Ms for mesyl or --SO2--CH3;
Ms2O for methanesulfonic anhydride or mesyl-anhydride;
NaN(TMS)2 for sodium bis(trimethylsilyl)amide; NaCl for sodium
chloride; NaH for sodium hydride; NaHCO3 for sodium bicarbonate or
sodium hydrogen carbonate; Na2CO3 sodium carbonate; NaOH for
sodium hydroxide; Na2SO4 for sodium sulfate; NaHSO3 for
sodium bisulfite or sodium hydrogen sulfite; Na2S2O3 for
sodium thiosulfate; NH2NH2 for hydrazine; NH4HCO3 for
ammonium bicarbonate; NH4Cl for ammonium chloride; NMMO for
N-methylmorpholine N-oxide; NaIO4 for sodium periodate; Ni for
nickel; OH for hydroxyl; OsO4 for osmium tetroxide; TBAF for
tetrabutylammonium fluoride; TEA or Et3N for triethylamine; TFA for
trifluoroacetic acid; THF for tetrahydrofuran; TMEDA for
N,N,N',N'-tetramethylethylenediamine; TPP or PPh3 for
triphenyl-phosphine; Troc for 2,2,2-trichloroethyl carbonyl; Ts for tosyl
or --SO2--C6H4--CH3; Ts2O for tolylsulfonic
anhydride or tosyl-anhydride; TsOH for p-tolylsulfonic acid; Pd for
palladium; Ph for phenyl; POPd for dihydrogen
dichlorobis(di-tert-butylphosphinito-KP)palladate(II);
Pd2(dba)3 for tris(dibenzylideneacetone) dipalladium (0);
Pd(PPh3)4 for tetrakis(triphenylphosphine)palladium (0);
PdCl2(PPh3)2 for
trans-dichlorobis-(triphenylphosphine)palladium (II); Pt for platinum; Rh
for rhodium; Ru for ruthenium; TBS for tert-butyl dimethylsilyl; TMS for
trimethylsilyl; or TMSC1 for trimethylsilyl chloride.

Synthetic Methods

[0202] The compounds and processes of the present invention will be better
understood in connection with the following synthetic schemes that
illustrate the methods by which the compounds of the invention may be
prepared. These schemes are of illustrative purpose, and are not meant to
limit the scope of the invention. Equivalent, similar, or suitable
solvents, reagents or reaction conditions may be substituted for those
particular solvents, reagents, or reaction conditions described herein
without departing from the general scope of the method of synthesis.

[0204] General procedure to prepare 2'-chloroacetylenyl nucleoside of
present invention is depicted in scheme 1. The ketone compound (1-2),
herein P1, P2 and P3 are hydroxyl protection group, such
as but not limited to, benzoyl, acetyl, TMS, TIPS, was made from
2-hydroxy compound (1-1) according to the modified procedure from the
account by Cook, G. P; Greenberg, M. M. J. Org. Chem. 1994, 59, 4704. The
addition of chloroacetylene to ketone has also been well described in the
patent, U.S. Pat. No. 3,290,297 by Cross, A. D. The ketone compound (1-2)
was treated with the intermediate generated from 1,2-dichloroethylene and
methyllithium in the presence of metal halides such as, but not limited
to, CeCl3, in aprotic solvent such as diethyl ether, THF, DME,
followed by protection of resulting 2'-hydroxy group with P4, such
as but not limited to, benzoyl, acetyl, TMS, TIPS to give the
chloroacetylenyl compound (1-3) in good yield.

##STR01289##

[0205] A more thorough discussion of the procedures, reagents and
conditions for protecting hydroxyl group is described in literature, for
example, by T. W. Greene and P. G. M. Wuts in "Protective Groups in
Organic Synthesis" 3rded., John Wiley & Son, Inc., 1999.

[0206] Further glycosylation of the nucleobase (1-3) was accomplished by
treatment of the compound (1-3) with the appropriate persilylated
nucleobase in the presence of a Lewis acid such as, but not limited to,
SnCl4, TiCl4, TMSOTf, HgO or HgBr2 in an aprotic solvent
such as, but not limited to, toluene, acetonitrile, benzene, THF, DME, or
a mixture of any or all of these solvents. The reaction temperature was
from room temperature to 150° C. The optional protecting groups in
the protected nucleosides or the compound formula (1-4) can be cleaved
following established deprotection methodologies. A more thorough
discussion of the procedures, reagents and conditions for deprotection is
described in literature, for example, by T. W. Greene and P. G. M. Wuts
in "Protective Groups in Organic Synthesis" 3rded., John Wiley & Son,
Inc., 1999.

[0209] It will be appreciated that, with appropriate manipulation and
protection of any chemical functionality, synthesis of compounds of
Formula (I) is accomplished by methods analogous to those above and to
those described in the Experimental section. Suitable protecting groups
can be found, but are not restricted to, those found in T W Greene and P
G M Wuts "Protective Groups in Organic Synthesis", 3rd Ed (1999), J Wiley
and Sons.

[0213] To a mixture of 1.3.5-tri-O-benzoyl-α-D-ribifuranose (23.58
g, 50.99 mmol) (1) in dry CH2Cl2 (250 mL) was portionwise added
Dess-Martin periodinane (30.8 g, 72.4 mmol) at 0° C. and stirred
for 15 min. Then, it was allowed to warm to room temperature and stirred
for 17 hrs. The reaction mixture was evaporated off, triturated with
diethyl ether (360 mL) and filtered through a fritted funnel. The
filtrate was treated with sodium thiosulfate (10 g) and saturated
NaHCO3 aqueous solution (400 mL) and stirred vigorously for 15 min.
It was separated and the aqueous layer was extracted with diethyl ether
(100 mL). The combined organic layer was washed with H2O and brine,
dried over Na2SO4, filtered and evaporated to dryness. The
residue was azeothropically dried with dry CH2Cl2, toluene and
hexanes successively to give white foam (2) (22.8 g). MS: (ESI-MS) m/z
(M+H) 461.12.

Step 1b:

##STR01292##

[0215] To a mixture of cis-1,2-dichloroethylene (3.8 mL, 50.34 mmol) in
dry diethyl ether (80 mL) was dropwise added 1.6M methyllithium in
diethyl ether (31.7 mL, 50.66 mmol) at 0° C. and stirred for 10
min. The reaction mixture was allowed to warm to room temperature and
stirred for 105 min. Then, it was cooled to -78° C. and
transferred to CeCl3 (dried, 29 g) in dry diethyl ether (pre-cooled
at -78° C.) via cannula and stirred at -78° C. for ˜2
hrs before the addition of the product of step 1a (2) (7.29 g, 15.83
mmol) in dry diethyl ether (50 mL). The reaction mixture was stirred at
-78° C. for 1 hr and quenched by the addition of saturated
NH4Cl aqueous solution (200 mL), allowed to warm to room
temperature, filtered through a pad of celite, washed with saturated
NH4Cl aqueous solution and methyl tert-butyl ether (200 mL) and
separated. The aqueous layer was extracted with ethyl acetate (200 mL).
The combined organic layer was washed with H2O and brine, dried over
Na2SO4, filtered and evaporated to give the chloroacetylene
adduct as a white foam (7.625 g), which was directly used for
benzoylation without purification. To a mixture of the chloroacetylene
adduct (7.62 g, 14.628 mmol), DMAP (1.787 g, 14.628 mmol) and
triethylamine (12.23 mL, 87.77 mL) in dry CH2Cl2 (150 mL) was
added benzoyl chloride (5.09 mL, 43.88 mmol) at 0° C. and stirred
for 7 min. Then, it was allowed to warm to room temperature and stirred
for 3 hrs. The reaction was quenched by the addition of methanol (10 mL),
stirred for 10 min, diluted with CH2Cl2 (200 mL), washed with
1M-HCl, saturated NaHCO3 aqueous solution and brine, successively.
The organic layer was dried over Na2SO4, filtered and
evaporated to dryness. The residue was purified by silica gel column
chromatography with 0˜25% ethyl acetate in hexanes to give the
compound (3) (7.904 g, α:β=1:3). MS: (ESI) m/z (M+Na) 647.09.

Step 1c:

##STR01293##

[0217] A mixture of uracil (2.572 g, 22.94 mmol) and
N,O-bis(trimethylsilyl)acetamide (11.2 mL, 45.89 mmol) in dry
acetonitrile (50 mL) was refluxed for 30 min and cooled to room
temperature. The product of step 1b (3) in dry acetonitrile (70 mL) was
transferred to previously made bis-silylenolether via cannula. Then,
SnCl4 (5.8 mL, 49.33 mmol) was added to the reaction mixture and
refluxed for 5.5 hrs. After cooling to 0° C., the resction mixture
was diluted with ethyl acetate (300 mL), slowly treated with cold
saturated NaHCO3 aqueous solution (350 mL), stirred for 20 min and
filtered through a pad of celite. The filtrate was separated. The aqueous
layer was extracted with ethyl acetate (2×100 mL). The combined
organic layer was washed with saturated NaHCO3 aqueous solution and
brine, successively. The organic layer was dried over Na2SO4,
filtered and evaporated to dryness. The residue was purified by silica
gel column chromatography with 0˜60% ethyl acetate in hexanes to
give the compound (4) (4.93 g) as a white foam. MS: (ESI) m/z (M+H)
615.04.

Step 1d:

##STR01294##

[0219] A mixture of the product of step 1c (4) (5.51 g, 8.96 mmol) in 7N
ammonia in methanol was stirred at room temperature for 16 hrs and
evaporated to dryness. The residue was purified by silica gel column
chromatography with 0˜10% MeOH in CH2Cl2 to afford the
title compound (2.093 g) as a white foam. MS: (ESI) m/z (M+H) 303.02.

[0222] To a mixture of example 1 (25 mg) and phosphochloridate 5a (5 eq.)
in dry THF (2˜3 mL) was added 1-methylimidazole (6 eq.) at
0° C., allowed to warm to room temperature and stirred for 15 hrs
before the addition of methanol (˜0.2 mL). After evaporation, the
residue was passed through a short silica gel column with 0˜6%
methanol in CH2Cl2. The collected fraction was evaporated and
purified HPLC using 15-90% acetonitrile in water as a mobile phase. The
combined fraction was evaporated in vacuo and lyophilized to afford the
title compound of example 2 as a white solid. MS: (ESI) m/z (M+H) 650.27.

[0225] The compound of example 3 was prepared by reaction the compound of
example 1 with compound of (5b) following the same precedure in the
preparation of the compound of example 2. MS: (ESI) m/z (M+H) 594.15.

[0228] The compound of example 4 was prepared by reaction the compound of
example 1 with compound of (5c) following the same precedure in the
preparation of the compound of example 2. MS: (ESI) m/z (M+H) 636.24.

[0231] The compound of example 5 was prepared by reaction the compound of
example 1 with compound of (5d) following the same precedure in the
preparation of the compound of example 2. MS: (ESI) m/z (M+H) 670.23.

[0234] The compound of example 6 was prepared by reaction the compound of
example 1 with compound of (5e) following the same precedure in the
preparation of the compound of example 2. MS: (ESI) m/z (M+H) 650.01.

[0237] The compound of example 7 was prepared by reaction the compound of
example 1 with compound of (5f) following the same precedure in the
preparation of the compound of example 2. MS: (ESI) m/z (M+H) 622.27.

[0240] The compound of example 8 was prepared by reaction the compound of
example 1 with compound of (5 g) following the same precedure in the
preparation of the compound of example 2. MS: (ESI) m/z (M+H) 648.26.

[0243] The compound of example 9 was prepared by reaction the compound of
example 1 with compound of (5h) following the same precedure in the
preparation of the compound of example 2. MS: (ESI) m/z (M+H) 662.28.

Biological Activity

1. HCV Replicon Cell Lines

[0244] HCV replicon cell lines (kindly provided by R. Bartenschlager)
isolated from colonies as described by Lohman et al. (Lohman et al.
(1999) Science 285: 110-113, expressly incorporated by reference in its
entirety) and used for all experiments. One of the HCV replicon cell
lines (strain Conl, genotype 1b) has the nucleic acid sequence set forth
in EMBL Accession No.: AJ242651, the coding sequence of which is from
nucleotides 1801 to 8406. Another replicon cell line (strain H77,
genotype 1a) was constructed as described by Yi et. al. (Yi et. al.
(2004) Journal of Virology 78(15):7904-15). The coding sequences of the
published HCV replicons were synthesized and subsequently assembled in
plasmids using standard molecular biology techniques.

[0247] EC50 values of single agent compounds were determined by HCV
RNA detection using quantitative RT-PCR, according to the manufacturer's
instructions, with a TAQMAN® One-Step RT-PCR Master Mix Reagents Kit
(Cat#AB 4309169, Applied Biosystems) on an ABI Model 7500 thermocycler.
EC50 values of combinations are similarly determined by HCV RNA
detection using quantitative RT-PCR. The TAQMAN primers to use for
detecting and quantifying HCV RNA obtained from Integrated DNA
Technologies. HCV RNA is normalized to GAPDH RNA levels in drug-treated
cells, which is detected and quantified using the Human GAPDH Endogenous
Control Mix (Applied Biosystems, AB 4310884E). Total cellular RNA is
purified from 96-well plates using the RNAqueous 96 kit (Ambion,
Cat#AM1812). Chemical agent cytotoxicity is evaluated using an MTS assay
according to the manufacturer's directions (Promega).

[0248] The compounds of the present invention can be effective against the
HCV 1a and 1b genotypes. It should also be understood that the compounds
of the present invention can inhibit multiple genotypes of HCV. In one
embodiment, compounds of the present invention are active against the 1a,
1b, 2a, 2b, 3a, 4a, and 5a genotypes. Table 6 shows the EC50 values
of representative compounds of the present invention against the HCV 1a
and 1b genotypes from the above described qRT-PCR. EC50 ranges
against HCV 1a or 1b are as follows: A>1 μM; B 0.1-1 μM;
C<0.1 μM.

[0249] While this invention has been particularly shown and described with
references to preferred embodiments thereof, it will be understood by
those skilled in the art that various changes in form and details may be
made therein without departing from the scope of the invention
encompassed by the appended claims.